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Current Research and Scholarly Interests

The overall goal of our research is to understand on both a molecular and systemic level how hormones regulate stem cell fate decisions and the role these pathways play in both physiology and disease. We use molecular biology and in vivo models to elucidate mechanisms of regulating cell fate determination by the endocrine system. Understanding these processes has profound and broad implications for both science and health.

Journal Articles

Abstract

The increased incidence of obesity and metabolic disease underscores the importance of elucidating the biology of adipose tissue development. The recent discovery of cell surface markers for prospective identification of adipose precursor cells (APCs) in vivo will greatly facilitate these studies, yet tools for specifically targeting these cells in vivo have not been identified. Here, we survey three transgenic mouse lines, Fabp4-Cre, PdgfRα-Cre, and Prx1-Cre, precisely assessing Cre-mediated recombination in adipose stromal populations and mature tissues. Our data provide key insights into the utility of these tools to modulate gene expression in adipose tissues. In particular, Fabp4-Cre is not effective to target APCs, nor is its activity restricted to these cells. PdgfRα-Cre directs recombination in the vast majority of APCs, but also targets other populations. In contrast, adipose expression of Prx1-Cre is chiefly limited to subcutaneous inguinal APCs, which will be valuable for dissection of APC functions among adipose depots.

Abstract

Type 1 diabetes (T1D) is an autoimmune disease, whereas type 2 diabetes (T2D) results from insulin resistance and beta cell dysfunction. Previously, the onset of these two separate diseases was easily distinguished, with children being most at risk for T1D and T2D occurring in overweight adults. However, the dramatic rise in obesity, coupled with the notable increase in T1D, has created a large overlap in these previously discrete patient populations. Delayed diagnosis of T1D can result in severe illness or death, and rapid diagnosis of T1D is critical for the efficacy of emerging therapies. However, attempts to apply next-generation platforms have been unsuccessful for detecting diabetes biomarkers. Here we describe the development of a plasmonic gold chip for near-infrared fluorescence-enhanced (NIR-FE) detection of islet cell-targeting autoantibodies. We demonstrate that this platform has high sensitivity and specificity for the diagnosis of T1D and can be used to discover previously unknown biomarkers of T1D.

Abstract

Vitamin D is not really a vitamin but the precursor to the potent steroid hormone calcitriol, which has widespread actions throughout the body. Calcitriol regulates numerous cellular pathways that could have a role in determining cancer risk and prognosis. Although epidemiological and early clinical trials are inconsistent, and randomized control trials in humans do not yet exist to conclusively support a beneficial role for vitamin D, accumulating results from preclinical and some clinical studies strongly suggest that vitamin D deficiency increases the risk of developing cancer and that avoiding deficiency and adding vitamin D supplements might be an economical and safe way to reduce cancer incidence and improve cancer prognosis and outcome.

Abstract

White adipose tissue stores energy in the form of lipids, and brown adipose tissue expends energy via uncoupled fatty acid oxidation, which leads to the generation of heat. Obesity reflects an imbalance between energy storage and energy expenditure and is strongly associated with metabolic and cardiovascular disease. Therefore, there are important medical and biological implications for elucidating the mechanisms that promote energy expenditure in humans. Animal models with altered vitamin D receptor (VDR) expression have changes in energy expenditure. However, the specific mechanism for this effect has not been elucidated and the relevance for humans is unclear. Here we show, using human patient samples from individuals with hereditary vitamin D resistant rickets, that the VDR directly inhibits the expression of uncoupling protein-1 (UCP1), the critical protein for uncoupling fatty acid oxidation in brown fat and burning energy. The inhibition is enforced by VDR occupancy of a negative response element in the promoter proximal region of the UCP1 gene. Deletion of VDR increases UCP1 expression and results in a "browning" of adipocytes. Importantly, we found that this process occurs cell autonomously and is independent of the physiologic VDR hormone ligand, 1,25-dihydroxyvitamin D. These results identify a mechanism for modulating energy balance in humans.

Abstract

Glucocorticoids rapidly and robustly induce cell fate decisions in various multipotent cells, although the precise mechanisms of these important cellular events are not understood. Here we showed that glucocorticoids repressed Per3 expression and that this repression was critical for advancing mesenchymal stem cells to the adipocyte fate. Exogenous expression of Per3 inhibited adipogenesis, whereas knocking out Per3 enhanced that fate. Moreover, we found that PER3 formed a complex with PPAR? and inhibited PPAR?-mediated transcriptional activation via Ppar? response elements. Consistent with these findings, Per3 knock-out mice displayed alterations in body composition, with both increased adipose and decreased muscle tissue compared with wild-type mice. Our findings identify Per3 as potent mediator of cell fate that functions by altering the transcriptional activity of PPAR?.

Abstract

Circadian clock genes are regulated by glucocorticoids; however, whether this regulation is a direct or secondary effect and the physiological consequences of this regulation were unknown. Here, we identified glucocorticoid response elements (GREs) at multiple clock genes and showed that 3 were directly regulated by the glucocorticoid receptor. We determined that a GRE within the core clock gene Per2 was continuously occupied during rhythmic expression and essential for glucocorticoid regulation of that gene in vivo. We further demonstrated that mice with a genomic deletion spanning this GRE expressed elevated leptin levels and were protected from glucose intolerance and insulin resistance on glucocorticoid treatment but not from muscle wasting. We conclude that Per2 is an integral component of a particular glucocorticoid regulatory pathway and that glucocorticoid regulation of the peripheral clock is selectively required for some actions of glucocorticoids.

Abstract

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a common cause of hyponatremia. We describe two infants whose clinical and laboratory evaluations were consistent with the presence of SIADH, yet who had undetectable arginine vasopressin (AVP) levels. We hypothesized that they had gain-of-function mutations in the V2 vasopressin receptor (V2R). DNA sequencing of each patient's V2R gene (AVPR2) identified missense mutations in both, with resultant changes in codon 137 from arginine to cysteine or leucine. These novel mutations cause constitutive activation of the receptor and are the likely cause of the patients' SIADH-like clinical picture, which we have termed "nephrogenic syndrome of inappropriate antidiuresis."

Abstract

Diabetes mellitus is a common disease with a rising incidence and the findings of hyperglycemia and glucosuria. However, there are multiple types of diabetes, each with distinct etiologies. The two major types of diabetes are type 1, which is caused by an autoimmune process, and type 2, which is thought to be primarily metabolic, resulting from insulin resistance, often in the setting of obesity. Historically, the distinction between these two types was obvious. Here, we discuss how this paradigm has dramatically changed because of both the evolving epidemiology of diabetes mellitus and new and emerging tools, and therapies to diagnose and treat diabetes. As we believe that understanding these changes is critical to providing optimal care to patients with diabetes, we have developed a novel plasmonic gold chip platform that is able to meet the new and emerging demands of modern diabetes care.

Abstract

Patients with pancreatic agenesis are born without a pancreas, causing permanent neonatal diabetes and pancreatic enzyme insufficiency. These patients require insulin and enzyme replacement therapy to survive, grow, and maintain normal blood glucose levels. Pancreatic agenesis is an uncommon condition but high-throughput sequencing methods provide a rare opportunity to identify critical genes that are necessary for human pancreas development. Here we present the clinical history, evaluation, and the genetic and molecular analysis from two patients with pancreatic agenesis. Both patients were born with intrauterine growth restriction, minor heart defects and neonatal diabetes. In both cases, pancreatic agenesis was confirmed by imaging studies. The patients are clinically stable with pancreatic enzymes and insulin therapy. In order identify the etiology for their disease, we performed whole exome sequencing on both patients. For each proband we identified a de novo heterozygous mutation in the GATA6 gene. GATA6 is a homeobox containing transcription factor involved in both early development of the pancreas and heart. In vitro functional analysis of one of the variants revealed that the mutation creates a premature stop codon in the coding sequence resulting in the production of a truncated protein with loss of activity. These results show how genetic mutations in GATA6 may lead to functional inactivity and pancreatic agenesis in humans.

Abstract

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.

Abstract

Abstract A central clock consisting of interconnected positive and negative feedback gene loops operates in the brain, tying rhythmic activity to the 24-h day. The central clock entrains similar feedback loops present in most peripheral tissues to coordinate metabolic gene expression among organs and with feeding activity for more efficient utilization of resources. Recent studies are beginning to elucidate the intricate feedback mechanisms among central and peripheral clocks and their roles in activity and metabolic homeostasis. Adipose tissue serves as a major energy storage organ and releases paracrine and endocrine hormones to signal energy status to other organs. Within the adipose tissue, the transcriptional feedback regulation between clock genes and nuclear hormone receptors, together with direct protein associations among these molecules, ensures the expression of metabolic genes at the appropriate time. This review will summarize the important components and mechanisms of adipose clock entrainment, particularly highlighting instructive studies carried out in mice. This research not only illustrates the intricate connections between clocks and metabolism but also provides potential mechanisms to correct abnormalities induced by disrupted sleep or poor diet.

Abstract

The bioluminescence resonance energy transfer (BRET) technique has become extremely valuable for the real-time monitoring of protein-protein interactions in live cells. This method is highly amenable to the detection of G protein-coupled receptor (GPCR) interactions with proteins critical for regulating their function, such as ?-arrestins. Of particular interest to endocrinologists is the ability to monitor interactions involving endocrine receptors, such as orexin receptor 2 or vasopressin type II receptor. The BRET method utilizes heterologous co-expression of fusion proteins linking one protein of interest (GPCR) to a bioluminescent donor enzyme, a variant of Renilla luciferase, and a second protein of interest (?-arrestin) to an acceptor fluorophore. If in close proximity, energy resulting from oxidation of the coelenterazine substrate by the donor will transfer to the acceptor, which in turn fluoresces. Using novel luciferase constructs, we were able to monitor interactions not detectable using less sensitive BRET combinations in the same configuration. In particular, we were able to show receptor/?-arrestin interactions in an agonist-independent manner using Rluc8-tagged mutant receptors, in contrast to when using Rluc. Therefore, the enhanced BRET methodology has not only enabled live cell compound screening as we have recently published, it now provides a new level of sensitivity for monitoring specific transient, weak or hardly detectable protein-protein complexes, including agonist-independent GPCR/?-arrestin interactions. This has important implications for the use of BRET technologies in endocrine drug discovery programs as well as academic research.

Abstract

Nephrogenic syndrome of inappropriate antidiuresis is a recently identified genetic disease first described in two unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. It was found that each infant had a different mutation of the vasopressin type II receptor (V2R) at codon 137 where arginine was converted to cysteine or leucine (R137C or R137L), resulting in constitutive signaling. Interestingly, a missense mutation at the same codon, converting arginine to histidine (R137H), leads to the opposite disease phenotype with a loss of the kidney's ability to concentrate urine resulting in nephrogenic diabetes insipidus. This mutation is associated with impaired signaling, although whether this is predominantly due to impaired trafficking to the plasma membrane, agonist-independent internalization, or G protein uncoupling is currently unclear. Using bioluminescence resonance energy transfer and confocal microscopy, we demonstrate that both V2R-R137C and V2R-R137L mutants interact with beta-arrestins in an agonist-independent manner resulting in dynamin-dependent internalization. This phenotype is similar to that observed for V2R-R137H, which is intriguing considering that it is accompanied by constitutive rather than impaired signaling. Consequently, it would seem that agonist-independent internalization per se is unlikely to be the major determinant of impaired V2R-R137H signaling. Our findings indicate that the V2R-R137C and V2R-R137L mutants traffic considerably more efficiently to the plasma membrane than V2R-R137H, identifying this as a potentially important mutation-dependent difference affecting V2R function.

Abstract

Metabolic disease is a well established major public health problem in the adult population. However, the origins of metabolic disease of adults can begin early in life. In addition, in recent years, there has been a disturbing increase in the number of children developing the full presentation of metabolic disease as a result of the increase in obesity in this population. Therefore, pediatricians and pediatric physician-scientists are essential both for instituting preventive measures and developing new therapies. This challenge has been met with a substantial increase in research into both the clinical and basic science of metabolism. A connection between glucocorticoids and the origins of metabolic disease is one enticing clue because of the clinical similarity between patients with glucocorticoid excess and those with metabolic disease. This perspective highlights one series of investigations that has advanced our understanding of the development of metabolic disease. In this work, a unifying link was found by investigating the role of glucocorticoids on cell fate and differentiation of mesenchymal stem cells. We conclude that elucidating the mechanisms by which glucocorticoids modulate cell fate decisions holds promise for developing new therapies and preventative measures.

Abstract

The glucocorticoid receptor (GR) interacts with specific GR-binding sequences (GBSs) at glucocorticoid response elements (GREs) to orchestrate transcriptional networks. Although the sequences of the GBSs are highly variable among different GREs, the precise sequence within an individual GRE is highly conserved. In this study, we examined whether sequence conservation of sites resembling GBSs is sufficient to predict GR occupancy of GREs at genes responsive to glucocorticoids. Indeed, we found that the level of conservation of these sites at genes up-regulated by glucocorticoids in mouse C3H10T1/2 mesenchymal stem-like cells correlated directly with the extent of occupancy by GR. In striking contrast, we failed to observe GR occupancy of GBSs at genes repressed by glucocorticoids, despite the occurrence of these sites at a frequency similar to that of the induced genes. Thus, GR occupancy of the GBS motif correlates with induction but not repression, and GBS conservation alone is sufficient to predict GR occupancy and GRE function at induced genes.

Abstract

The rise in the incidence of metabolic disease to become a major public health problem has been met with a substantial increase in research into both the clinical and basic science of metabolism. This work has revealed that the origins of metabolic diseases of adults can begin early in life. Furthermore, the age of onset of symptoms has been rapidly decreasing. Therefore, pediatricians should be critically involved in both the generation of new therapies as well as the institution of measures of disease prevention. This perspective examines how recent advances have improved our understanding of the development of metabolic diseases. A connection between glucocorticoids and the origins of metabolic disease is one enticing clue because of the clinical similarity between patients with glucocorticoid excess and those with metabolic disease. A unifying link was found by investigating the role of glucocorticoids on cell fate and differentiation of mesenchymal stem cells. We conclude that understanding the mechanisms by which glucocorticoids can modify how cell fate decisions are made holds promise for developing new therapies and preventative measures.

Abstract

Thyroid nodules are rare in children compared to adults. Although most thyroid nodules are benign, the risk of malignancy is greater in pediatric patients. Papillary and follicular cell tumors account for the majority of thyroid neoplasms; Hürthle cell tumors account for less than 5%. Despite being uncommon, malignant Hürthle cell tumors are potentially more aggressive than papillary and follicular cell tumors. Therefore, distinguishing between types of thyroid neoplasms in a timely fashion has implications for prognosis and therapy. We describe a 12-year-old peripubertal girl who presented with a large right-sided thyroid nodule that was subsequently diagnosed as a Hürthle cell adenoma. To our knowledge, she represents the youngest patient with a Hürthle cell neoplasm.

Abstract

Studies indicate that running a marathon can be associated with increases in serum cardiac troponin levels. The clinical significance of such increases remains unclear. We seek to determine the prevalence of troponin increases and epidemiologic factors associated with these increases in a large and heterogeneous cohort of marathon finishers.Entrants in the 2002 Boston Marathon were recruited 1 to 2 days before the race. Data collected included demographic and training history, symptoms experienced during the run, and postrace troponin T and I levels. Simple descriptive statistics were performed to describe the prevalence of troponin increases and runner characteristics.Of 766 runners enrolled, 482 had blood analyzed at the finish line. In all, 34% were women, 20% were younger than 30 years, and 92% had run at least 1 previous marathon. Most runners (68%) had some degree of postrace troponin increase (troponin T > or = 0.01 ng/mL or troponin I > or = 0.1 ng/mL), and 55 (11%) had significant increases (troponin T > or = 0.075 ng/mL or troponin I > or = 0.5 ng/mL). Running inexperience (< 5 previous marathons) and young age (< 30 years) were associated with elevated troponins. These correlates were robust throughout a wide range of troponin thresholds considered. Health factors, family history, training, race performance, and symptoms were not associated with increases.Troponin increases were relatively common among marathon finishers and can reach levels typically diagnostic for acute myocardial infarction. Less marathon experience and younger age appeared to be associated with troponin increases, whereas race duration and the presence of traditional cardiovascular risk factors were not. Further work is needed to determine the clinical significance of these findings.

Abstract

Mutations of G protein-coupled receptors are responsible for a wide range of diseases. With respect to water balance and vasopressin signaling, more than 180 different inactivating mutations have been previously described in the V2 vasopressin receptor (V2R), resulting in nephrogenic diabetes insipidus. In contrast, we have recently described the first known patients with V2R activating mutations. Patients with these novel gain-of-function V2R mutations have a disorder which we have termed "nephrogenic syndrome of inappropriate antidiuresis" (NSIAD): a clinical presentation consistent with the syndrome of inappropriate antidiuretic hormone secretion but with undetectable levels of arginine vasopressin (antidiuretic hormone). The mechanisms by which these mutations constitutively activate the V2R are currently being investigated.

Myostatin modulates adipogenesis to generate adipocytes with favorable metabolic effectsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAFeldman, B. J., Streeper, R. S., Farese, R. V., Yamamoto, K. R.2006; 103 (42): 15675-15680

Abstract

A pluripotent cell line, C3H10T1/2, is induced to undergo adipogenesis by a mixture of factors that includes a glucocorticoid such as dexamethasone. We found that expression of myostatin (MSTN), a TGF-beta family member extensively studied in muscle, was induced by dexamethasone under those differentiation conditions. Moreover, MSTN could substitute for dexamethasone in the adipogenesis mixture. However, the adipocytes induced by MSTN in both cell culture and transgenic mice were small and expressed markers characteristic of immature adipocytes. These adipocytes exhibited cell-autonomous increases in insulin sensitivity and glucose oxidation. In mice, these effects produced elevated systemic insulin sensitivity and resistance to diet-induced obesity. Modulation of the final stages of adipogenesis may provide a novel approach to understanding and treating metabolic disease.

Abstract

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a common cause of hyponatremia. We report findings in 2 unrelated male infants whose clinical presentation and laboratory findings were consistent with SIADH, but who exhibited unmeasurable arginine vasopressin (AVP) levels on repeated occasions. We hypothesized that these infants had a novel gain of function defect in the AVP-signaling pathway. DNA sequencing of each patient's vasopressin V2 receptor (V2R) gene identified mutations (R137C or R137L) in each. R137H mutations have been reported previously to cause nephrogenic diabetes insipidus. To further characterize the effects of these mutations, we re-created each mutation by site-directed mutagenesis in a vasopressin V2R expression vector and cotransfected COS-7 cells with wild-type and mutant vasopressin V2R vectors and a cyclic adenosine monophosphate-responsive luciferase reporter plasmid. The luciferase activity was induced 7.5-fold (R137L mutant; P = 0.0037) and 4-fold (R137C mutant; P = 0.013) more than the wild-type vasopressin V2R, which is the empty vector or the inactivating R137H mutant. This novel gain of function mutation in the vasopressin V2R can cause constitutive activation of the receptor and resultant hyponatremia. These findings represent a previously unrecognized genetic disease, which was designated as nephrogenic syndrome of inappropriate antidiuresis. A number of questions have emerged, including the following: (1) What is the frequency? (2) Are there nonrenal manifestations? (3) Are heterozygotes affected? (4) What is the optimal therapy? and (5) How do these mutations cause constitutive activation of the receptor?

Abstract

We report the successful use of oral urea in the management of children with chronic syndrome of inappropriate antidiuretic hormone secretion (SIAD). We performed a retrospective review of four children with chronic SIAD. After initial attempts at management with fluid restriction, each was started on a 30% to 50% oral urea solution, and the dose was titrated until normal serum sodium was achieved. Fluid intake was liberalized after serum sodium normalization. All four children normalized their serum sodium. No side effects or toxicities were experienced. Oral urea is a safe, effective treatment for chronic SIAD in children.

Abstract

We describe an infant with a persistent pericardial effusion who was diagnosed with carbohydrate-deficient glycoprotein syndrome (CDGS)-Ia. She was born with mild dysmorphic features and common cardiac abnormalities. However, she re-presented at 2.5 months of age with a pericardial effusion. We decided to embark on a therapeutic trial of corticosteroids and salicylic acid therapy in an attempt to avoid pericardectomy. After 3 weeks of medical treatment the effusion resolved. This experience allows us to propose that medical management with corticosteroids and salicylic acid can be considered as an alternative to surgical therapy for CDGS-I patients with persistent pericardial effusions.

Abstract

The normal prostate and early-stage prostate cancers depend on androgens for growth and survival, and androgen ablation therapy causes them to regress. Cancers that are not cured by surgery eventually become androgen independent, rendering anti-androgen therapy ineffective. But how does androgen independence arise? We predict that understanding the pathways that lead to the development of androgen-independent prostate cancer will pave the way to effective therapies for these, at present, untreatable cancers.

Abstract

PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a Notch(IC) allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involving Notch1 occurred in a more 3' exon, resulting in truncating mutations that deleted the carboxy-terminal region of Notch1 containing negative regulatory sequences (Notch1(DeltaC)). In contrast to Notch(IC), forced expression of Notch1(DeltaC) in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1(DeltaC) genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation of Notch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development. (Blood. 2000;96:1906-1913)

Abstract

Mice transgenic for the leukemia oncogene E2A-PBX1 invariably develop lethal, high-grade T-cell lymphomas by 5 months of age. In this study, retroviral insertional mutagenesis was employed to identify oncogenes that cooperate with the E2A-PBX1 transgene in lymphomagenesis. Neonatal retroviral infection substantially reduced length of survival due to accelerated development of lymphomas (81 versus 130 days). The Pim1 gene was targeted by retroviral insertions in 48% of accelerated lymphomas whereas less than 5% contained activated c-Myc and none contained activated Pim2. However, Pim1 DNA rearrangements were frequently sub-stoichiometric and not present at all sites of involvement in an otherwise monoclonal lymphoma indicating that Pim1 activation occurred late in the course of lymphomagenesis. Tumor subpopulations containing activated Pim1 alleles displayed a substantial growth advantage over Pim1 negative cells following serial transfer to secondary, syngeneic recipients. Cooperative interactions were observed in intercrossed Pim1 and E2A-PBX1 transgenic mice in which all double transgenic progeny developed lethal, diffuse T lineage lymphomas by 3 months of age, whereas only 13% of E2A-PBX1 and none of Pim1 single transgenic intercross progeny developed lymphomas by 1 year. Tumors from double transgenic mice were monoclonal providing evidence that additional genetic events were required for transformation. Therefore, Pim1 and E2a-Pbx1 cooperate in T lineage lymphomagenesis but they are not sufficient and the role of Pim1 is more likely to be associated with tumor progression.

Abstract

Oncogenic mutation of nuclear transcription factors often is associated with altered patterns of subcellular localization that may be of functional importance. The leukemogenic transcription factor gene E2A-PBX1 is created through fusion of the genes E2A and PBX1 as a result of t(1;19) in acute lymphoblastic leukemia. We evaluated subcellular localization patterns of E2A-PBX1 protein in transfected cells using immunofluorescence. Full-length E2A-PBX1 was exclusively nuclear and was concentrated in spherical domains denoted chimeric-E2A oncoprotein domains (CODs). In contrast, nuclear fluorescence for wild-type E2A or PBX1 proteins was diffuse. Enhanced concentrations of RNA polymerase II within many CODs and the requirement for an E2A-encoded activation domain suggested transcriptional relevance. However, in situ co-detection of nascent transcripts labeled with bromouridine failed to confirm altered transcriptional activity in relation to CODs. CODs also failed to co-localize with other proteins known to occupy functional nuclear compartments, including the transcription factor PML, the spliceosome-associated protein SC-35 and the adenovirus replication factor DBP, or with foci of DNA replication. Co-transfection of Hoxb7, a homeodomain protein capable of enhancing DNA binding by PBX1, impaired COD formation, suggesting that CODs contain E2A-PBX1 protein not associated with DNA. We conclude that, as a 'gain of function' phenomenon requiring protein elements from both E2A and PBX1, COD formation may be relevant to the biology of E2A-PBX1 in leukemogenesis.

Abstract

DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.